The T cell receptor (TCR) is generated by rearrangement of V, D and J segments in individual cells at the genomic level. Therefore, the TCR repertoire collectively can recognize any antigen. As a consequence of this random rearrangement process, T cells lack the ability to discriminate between self antigens and pathogen- associated antigens. Instead, T cells must be activated by innate immune antigen presenting cells (APCs) and instructed to differentiate into effector cells. This activating signal requires antigen-dependent stimulation of the TCR and costimulation through CD28, which is a membrane receptor constitutively expressed on all naove T cells and subsets of memory T cells. Binding to its ligands B7.1 (CD80) or B7.2 (CD86) expressed on APCs activated by recognition of pathogen-associated molecules delivers a costimulatory signal that synergizes with TCR signaling and promotes cell survival, proliferation and effector functions. The mechanism by which ligand engagement by TCR or CD28 leads to receptor triggering and signal transduction remains unknown;a general mechanism of receptor triggering remains to be elucidated for any phosphotyrosine-based immune receptor. Conversely, the mechanism preventing spontaneous and aberrant signaling of CD28 and other phosphotyrosine-based receptors is also poorly understood. Using a combination of fluorescence resonance energy transfer (FRET) microscopy, biochemical and cellular assays to assess T cell signaling and function, and an in vivo model of multiple sclerosis (murine experimental autoimmune encephalomyelitis), this research will examine: a) association of CD28 cytoplasmic domain (CD28CD) with the inner leaflet of the plasma membrane;b) the consequences of this association for regulation of signaling and T cell function;and c) the mechanism by which CD28CD is released from the plasma membrane under physiological conditions. Mechanistic understanding of CD28 regulation would provide insight into a more generalizable model of receptor triggering.